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Hpy188I-DNA pre- and post-cleavage complexes--snapshots of the GIY-YIG nuclease mediated catalysis.

Sokolowska M, Czapinska H, Bochtler M - Nucleic Acids Res. (2010)

Bottom Line: In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine.A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site.This metal ion contacts the phosphate proS oxygen atom and the leaving group 3'-oxygen atom, presumably to facilitate its departure.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.

ABSTRACT
The GIY-YIG nuclease domain is present in all kingdoms of life and has diverse functions. It is found in the eukaryotic flap endonuclease and Holliday junction resolvase Slx1-Slx4, the prokaryotic nucleotide excision repair proteins UvrC and Cho, and in proteins of 'selfish' genetic elements. Here we present the structures of the ternary pre- and post-cleavage complexes of the type II GIY-YIG restriction endonuclease Hpy188I with DNA and a surrogate or catalytic metal ion, respectively. Our structures suggest that GIY-YIG nucleases catalyze DNA hydrolysis by a single substitution reaction. They are consistent with a previous proposal that a tyrosine residue (which we expect to occur in its phenolate form) acts as a general base for the attacking water molecule. In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine. A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site. This metal ion contacts the phosphate proS oxygen atom and the leaving group 3'-oxygen atom, presumably to facilitate its departure. Taken together, our data reveal striking analogy in the absence of homology between GIY-YIG and ββα-Me nucleases.

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GIY-YIG nuclease alignment and Hpy188I fold: (A) Representative GIY-YIG nucleases were aligned by a combination of structure- and sequence-based methods. The alignment was corrected manually to take into account mutagenesis data that have identified active site residues and previously published alignments (19,24,26). The numbering of conserved residues and secondary structure information in the first and second lines refer to Hpy188I. (B) A single Hpy188I subunit and the domain swapped region of the other subunit are shown in symbolic representation, with cylinders for α-helices and arrows for β-strands. Short 3/10 helices are omitted for clarity. The DNA is represented by the smoothed backbones (proximal strand in gray and distal strand in black).
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Figure 1: GIY-YIG nuclease alignment and Hpy188I fold: (A) Representative GIY-YIG nucleases were aligned by a combination of structure- and sequence-based methods. The alignment was corrected manually to take into account mutagenesis data that have identified active site residues and previously published alignments (19,24,26). The numbering of conserved residues and secondary structure information in the first and second lines refer to Hpy188I. (B) A single Hpy188I subunit and the domain swapped region of the other subunit are shown in symbolic representation, with cylinders for α-helices and arrows for β-strands. Short 3/10 helices are omitted for clarity. The DNA is represented by the smoothed backbones (proximal strand in gray and distal strand in black).

Mentions: Superpositions of the GIY-YIG nuclease structures reveal a conserved core that is present almost without additional ‘decorations’ in the UvrC and I-TevI. The ‘GIY-YIG hairpin’ anchors the GIY and YIG motifs, which are both subject to substantial variation. In the first motif (GIY), ‘Y’ is conserved, but ‘G’ and ‘I’ are present only in some enzymes. In the second motif (YIG), ‘Y’ is almost universal, but replaced by a lysine in Hpy188I, ‘I’ can be substituted with a valine, and ‘G’ is strongly conserved (1) The next core element is an α-helix which we name the ‘arginine helix’ because of an invariant arginine residue. This is followed by a ‘linker strand’, which extends the GIY-YIG hairpin to an antiparallel β-sheet. The last conserved secondary structure element is another α-helix that we call the ‘glutamate helix’ because of a glutamate residue that stands out in the alignment (Figure 1A).Figure 1.


Hpy188I-DNA pre- and post-cleavage complexes--snapshots of the GIY-YIG nuclease mediated catalysis.

Sokolowska M, Czapinska H, Bochtler M - Nucleic Acids Res. (2010)

GIY-YIG nuclease alignment and Hpy188I fold: (A) Representative GIY-YIG nucleases were aligned by a combination of structure- and sequence-based methods. The alignment was corrected manually to take into account mutagenesis data that have identified active site residues and previously published alignments (19,24,26). The numbering of conserved residues and secondary structure information in the first and second lines refer to Hpy188I. (B) A single Hpy188I subunit and the domain swapped region of the other subunit are shown in symbolic representation, with cylinders for α-helices and arrows for β-strands. Short 3/10 helices are omitted for clarity. The DNA is represented by the smoothed backbones (proximal strand in gray and distal strand in black).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045582&req=5

Figure 1: GIY-YIG nuclease alignment and Hpy188I fold: (A) Representative GIY-YIG nucleases were aligned by a combination of structure- and sequence-based methods. The alignment was corrected manually to take into account mutagenesis data that have identified active site residues and previously published alignments (19,24,26). The numbering of conserved residues and secondary structure information in the first and second lines refer to Hpy188I. (B) A single Hpy188I subunit and the domain swapped region of the other subunit are shown in symbolic representation, with cylinders for α-helices and arrows for β-strands. Short 3/10 helices are omitted for clarity. The DNA is represented by the smoothed backbones (proximal strand in gray and distal strand in black).
Mentions: Superpositions of the GIY-YIG nuclease structures reveal a conserved core that is present almost without additional ‘decorations’ in the UvrC and I-TevI. The ‘GIY-YIG hairpin’ anchors the GIY and YIG motifs, which are both subject to substantial variation. In the first motif (GIY), ‘Y’ is conserved, but ‘G’ and ‘I’ are present only in some enzymes. In the second motif (YIG), ‘Y’ is almost universal, but replaced by a lysine in Hpy188I, ‘I’ can be substituted with a valine, and ‘G’ is strongly conserved (1) The next core element is an α-helix which we name the ‘arginine helix’ because of an invariant arginine residue. This is followed by a ‘linker strand’, which extends the GIY-YIG hairpin to an antiparallel β-sheet. The last conserved secondary structure element is another α-helix that we call the ‘glutamate helix’ because of a glutamate residue that stands out in the alignment (Figure 1A).Figure 1.

Bottom Line: In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine.A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site.This metal ion contacts the phosphate proS oxygen atom and the leaving group 3'-oxygen atom, presumably to facilitate its departure.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.

ABSTRACT
The GIY-YIG nuclease domain is present in all kingdoms of life and has diverse functions. It is found in the eukaryotic flap endonuclease and Holliday junction resolvase Slx1-Slx4, the prokaryotic nucleotide excision repair proteins UvrC and Cho, and in proteins of 'selfish' genetic elements. Here we present the structures of the ternary pre- and post-cleavage complexes of the type II GIY-YIG restriction endonuclease Hpy188I with DNA and a surrogate or catalytic metal ion, respectively. Our structures suggest that GIY-YIG nucleases catalyze DNA hydrolysis by a single substitution reaction. They are consistent with a previous proposal that a tyrosine residue (which we expect to occur in its phenolate form) acts as a general base for the attacking water molecule. In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine. A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site. This metal ion contacts the phosphate proS oxygen atom and the leaving group 3'-oxygen atom, presumably to facilitate its departure. Taken together, our data reveal striking analogy in the absence of homology between GIY-YIG and ββα-Me nucleases.

Show MeSH
Related in: MedlinePlus